Intrinsically disordered regions in nucleosome positioning barriers

核小体缺失启动子区域(核小体缺失启动子区域)和规则的核小体排列(定型的核小体排列模式)是真核生物中普遍存在的活性基因染色质模式，对转录调控是必不可少的，但仍不清楚。一般调节因子(GRF)，如酵母Abf1/Reb1或哺乳动物CTCF，不是经典的转录反式激活因子，但被认为能产生NDRs，并作为核小体屏障而形成阵列。事实上，我们证明了基本的NDR阵列模式是在Abf1/Reb1和ATP依赖的染色质重构体(如ISW2)的最小系统中重组的。现在，我们问是什么让GRF成为核小体屏障。除DNA结合区外，GRF在固有无序区(IDR)中高度富含。我们假设特定的IDR属性是GRF核小体屏障功能的关键。为了研究这一点，我们与罗希特·帕普教授(华盛顿大学，圣路易斯)合作，他们在IDR的生物物理学和理性IDR设计方面拥有丰富的经验，以询问功能。Pappu组的第一序列分析已经显示了GRFS的特征和保守的IDR组织，即DNA结合域两侧的长酸性IDR，这与经典反式激活子中的IDR组织不同。这为研究IDR的哪些特性通过a)随机突变(遗传筛选)；b)结构域互换；c)比较基因组学；以及d)根据IDR生物物理学进行从头设计提供了一个概念框架。我们将在体内测试屏障功能，并在我们独特的重建系统中通过直接生化评估进行测试。这样，我们将定义核小体屏障功能的具体分子要求。

Nucleosome depleted promoter regions (NDRs) followed by regular nucleosomal arrays (stereotypic NDR-array patterns) are the universal chromatin pattern of active genes in eukaryotes and are essential for transcription regulation, but remain unexplained. General regulatory factors (GRFs), like yeast Abf1/Reb1 or maybe mammalian CTCF, are not classical transactivators of transcription but suggested to generate NDRs and function as nucleosome barriers for array formation. Indeed, we showed that basic NDR-array patterns are reconstituted in a minimal system of Abf1/Reb1 and ATP dependent chromatin remodelers, like ISW2. Now, we ask what makes a GRF a nucleosome barrier. Besides DNA binding domains, GRFs are highly enriched in intrinsically disordered regions (IDRs). We hypothesize that specific IDR properties are key to the GRF nucleosome barrier function. To study this, we collaborate with the group of Prof. Rohit Pappu (Washington University, St. Louis) that has extensive experience in the biophysics of IDRs and in rational IDR design to interrogate function. First sequence analyses in the Pappu group already showed a characteristic and conserved IDR organization of GRFs, i.e., long acidic IDRs flanking the DNA binding domain, that are distinct from IDR organizations in classical transactivators. This provides a conceptional framework to study which IDR properties confer nucleosome barrier function by a) random mutagenesis (genetic screens); b) domain swaps; c) comparative genomics; and d) de novo design according to IDR biophysics. We will test for barrier function both in vivo as well as by direct biochemical assessment in our unique reconstitution system. This way we will define the specific molecular requirements for nucleosome barrier function.